Study On The New Technology Of Combinatorial Library Based On Modular Assembly

With the extensive application of genetic testing,immunochromatographic strips and other detection tools,biochemical testing attracts more and more attention.However,with the improvement of people's living standards,the traditional tool enzymes have been unable to adapt to people's demands.The directional evolution of protein is the most important measure for altering the structure and properties of enzymes in a short time.With continuous evolution in vitro,it is possible to gain evolutionary enzymes which possess strong environmental tolerance and high catalytic activity.The directed evolution of protein has been successfully applied to evolve of hundreds of enzymes,and have a huge impact on industry and daily life.There are two main measures of directional evolution of proteins.One method is based on the principle of gene mutation,such as Error-prone PCR,with the advantage of diversity of protein,but this method owns low efficiency and it is unable to use low homologous DNA fragments.The other one is based on the principle of gene recombination to build a protein library,such as DNA shuffling.This method can not only combine the low homologous DNA fragments,but also the non-homologous gene,this method greatly expands the range of DNA recombination,and has the advantages of highly efficiency,but the diversity decrease.In view of the advantages and disadvantages of the existing methods of building the protein library,This paper chose kanamycin resistance protein as our research object and designed a new technology of combinatorial library based on modular assembly.This paper use the secondary structure which was extracted from the original protein as a module,designing Linker which can be digested by Hinf I restriction endonuclease.The DNA template was positive connected and recombinant DNA fragment was amplified by PCR.Then,the combinatorial library was obtained by the connection,transformation,and screening with SV supporter made by our lab.After screening,10~5 positive clone were obtained.Through the electrophoretic analysis of 500 clone,it was known that the recombinant DNA fragments are different in size and have a good variety;By comparing the 20 cloning sequence,it is totally different from the wild type protein,and each module were all positive connection,indicated that the library had good diversity and high efficiency.Through the statistical analysis of the length and the different component of insertion fragment,length of different secondary structure module connection probability and the connection probability of module associated,fully illustrated the randomness of the module connection of constructed library.This paper have developed a new ligation method with random,simple and forward direction to construct the combinatorial library.This method did not place constraints on modules and enabled random assembly of many DNA parts into DNA constructs by a convenient workflow.In theory,a combinatorial library of M^N(M=Number of modules assembled,N=Number of modules excluding initiator and terminator)sequences should be achieved by our method.Owing to random assembly of DNA modules,our method could offer great insights for the structure-function relationships of the evolved proteins,and contribute to explore the sequence-structure protein landscape.Moreover,the constructed random combinatorial library may be very useful for high throughout screening and the computer simulation of protein,and the combinatorial construction of metabolic networks and pathways in synthetic biology.